Tuberous Sclerosis Complex (TSC) is a genetic disease caused by mutation of the TSC1 or TSC2 genes. Patients frequently have severe CNS manifestations including epilepsy, intellectual disabilities and autism. Cortical malformations (tubers) in the brains of patients with TSC are generally accepted as responsible for these severe neurological manifestations. Previous studies using human tissues and transgenic mouse and zebrafish models have led our focus on neural progenitor/stem cells. Abnormalities of neural progenitor cells may readily explain the increased cell number, atypical differentiation and ectopic position of neurons seen within the brain of patients with TSC. Several signaling pathways are known to be dysregulated in TSC though control of the mTOR kinase appears most critical. Recent findings support abnormalities of mTOR within two distinct protein complexes, mTORC1 and mTORC2. These alterations differ as we have found that Tsc1- deficient mouse neural progenitor cells appear to have increased mTORC1 but decreased mTORC2 signaling. However, the relative contribution of mTORC1 and mTORC2 signaling during the pathogenesis of TSC remains poorly understood. The overarching goal of this proposal is to determine the role of mTORC1 and mTORC2 signaling in the pathogenesis of TSC. This goal directly addresses the NINDS Strategic Plan by seeking to increase our knowledge of how the normal brain and nervous system develop, how these processes are subverted in disease and whether this information can lead to better treatments of neurological disorders. To achieve this goal we will use both transgenic mouse models featuring the inactivation of the mouse Tsc1 gene as well as induced pluripotent stem cells (iPSC) we have generated from patients with TSC due to loss of function mutations of the TSC1 or TSC2 genes. The direct comparison of mouse and human neural progenitor cells is a key aspect of this proposal as fundamental differences in the control of mTOR signaling in the brain likely arose during evolution. The Specific Aims of this project are to 1) Determine if TSC1/2-mutant human and Tsc1-deficient mouse neural progenitor cells generate excessive numbers of neurons; 2) Determine if neurons generated from TSC1-deficient and TSC2-deficient human and Tsc1-deficient mouse neural progenitors are able to acquire lower or upper layer identity and whether inhibition of mTORC1 signaling can restore these identities; 3). Determine if decreased mTORC2 signaling alone is sufficient to cause abnormal differentiation of mouse and human neural progenitor cells. These studies should greatly increase our understanding of the function of the mouse Tsc1 and human TSC genes. Our proposal is expected to define abnormal mTORC1 and mTORC2 signaling pathways in mouse and human cells and lead to the development of much more effective therapies for patients with TSC.